1. Field of the Invention (Technical Field)
The present invention relates to methods, compositions and devices for the detection of nucleic acids utilizing electrochemical detection with polymer-coated electrodes.
2. Background Art
The biological significance of nucleic acids has required the development of novel analytical methods for their detection and quantitation. Historical methods involve use of gel electrophoresis with radioactive labeled probes. Other label methods have been used, including biotin, digoxigenin and fluorescent dyes. These methods all involve long detection times and complex laboratory procedures, with ancillary amplification methods, such as polymerase chain reaction (PCR), frequently required to produce sufficient materials for detection.
In some applications, microscale separation techniques such as capillary electrophoresis are coupled to optical detection techniques for identification of individual nucleic acids. UV absorbance and laser-induced fluorescence systems have been used for this purpose. Electrochemical detectors, while used for other bioanalytical applications (Wang, J. Analytical Electrochemistry, VCH Publishers, New York, 1994; U.S. Pat. No. 5,516,644), have received only limited attention for nucleic acid analysis. However, electrochemical detector analysis systems, if demonstrated to be accurate and reproducible, offer potential advantages for detection of DNA and RNA and for xe2x80x9clab-on-a-chipxe2x80x9d devices, including high sensitivity and selectivity, ultra-small dead volumes, fast response, compatibility with advanced microfabrication and miniaturization technologies, low-cost, and minimal power requirements.
Electrochemical detection of DNA has traditionally relied on the electroactivity of nucleobases. In particular, the oxidation of the purine bases at carbon electrodes has been exploited for amperometric detection of nucleic acids in flow-injection (Wang, J.; Chen, L.; Chicharro, M. Anal. Chim. Acta, 319, (1996) 347) and capillary-electrophoresis (Xu, D. K.; Hua, L.; Chen, H. Y. Anal. Chim. Acta, 335, (1996) 95) systems. Another electrochemical route under evaluation relies on the oxidation of the sugar backbone at copper surfaces (Singhal, P.; Kuhr, W. G. Anal. Chem., 69, (1997) 4828). Such detection schemes can be used for monitoring both purine- and pyrimidine-containing nucleic acids, but require the use of an alkaline medium and specialized sinusoidal voltammetric instrumentation. In addition to these direct anodic detection schemes, several groups have explored indirect electrochemical protocols for detecting DNA (Takenaka, S.; Uto, Y.; Kondo, H.; Ihara, T.; Takagi, M. Anal. Biochem., 218, (1994) 436; Woolley, A. T.; Lao, K.; Glazer, A.; Mathies, R. A. Anal. Chem., 70, (1998) 684).
One application for biosensing devices is the in situ detection of DNA hybridization. Methods and devices for combining the base-pair recognition of DNA probes with the advantages of electrochemical transducers are currently receiving significant attention due to numerous potential applications (E. K. Wilson, Chem. Eng. News, May 25, 1998, 47; S. R. Mikkelsen, Electroanalysis 8(1996) 15). Most of these devices rely on measuring changes in the peak current of a redox-active marker that preferentially binds to the duplex formed in the hybridization event (K. M. Millan, S. R. Mikkelsen, Anal. Chem. 65(1993) 2317; K. Hashimoto, K. Ito, Y. Ishimori, Anal. Chem. 66(1994) 3830). Label-free electrochemical detection of hybridization reactions represents a very attractive approach for detecting DNA sequences (H. Korri-Youssoufi, F. Garnier, P. Srivastava, P. Godullot, A. Yassar, J. Am. Chem. Soc. 119(1997) 7388; P. Bauerle, A. Emge, Adv. Mater., 10(1998) 324; J. Wang, G. Rivas, J. Fernandes, L. Paz, M. Jiang, R. Waymire, Anal. Chim. Acta 375(1998) 197; and E. Souteyrand, J. P. Cloarec, J. R. Martin, C. Wilson, I. Lawrence, S. Mikkelsen, M. F. Lawrence, J. Phys. Chem. B 101(1997) 2980). Such approaches rely on monitoring changes in electronic or interfacial properties accompanying the DNA hybridization event. Label-free detection thus greatly simplifies the sensing protocol, since it eliminates the need for indicator addition, association and detection steps, and potentially offers instantaneous detection of the duplex formation.
Conducting-polymer molecular interfaces hold particular promise for inducing electrical signals resulting from DNA interactions. Changes in the properties of conducting polymers accompanying DNA hybridization have been reported in connection with the electropolymerization of oligonucleotide-substituted films (Korri-Youssoufi et al., supra; Bauerle et al., supra; U.S. Pat. No. 5,837,859). However, all such reported methods have been indirect, such as the method of Korri Youssoufi et al., which employs functionalized conjugated polymers, with an amino-substituted oligonucleotide grafted on a precursor copolymer, or the method of U.S. Pat. No. 5,837,859, employing copolymerization utilizing a covalent bond, such as with a spacer arm. Other methods, such as that of U.S. Pat. No. 5,156,810, employ a polymerized surfactant layer incorporating a ligand on a substrate. Other patents of interest include U.S. Pat. No. 5,776,672, which employs secondary substrates and requires approximately one hour per assay. The immobilization of DNA onto conductive surfaces is of enormous interest both in studies of DNA itself and numerous applications ranging from DNA diagnostics to gene therapy. A key requirement for such investigations and applications is the achievement of an efficient interface between the nucleic acid system and the conductive surface.
In one embodiment, the invention is an apparatus for detection of DNA hybridization, which apparatus includes a specimen electrode with a detection surface and a conducting polymer composition coating at least a portion of the detection surface of the specimen electrode, wherein the conducting polymer composition comprises a conducting polymer and a free oligomer complementary to the DNA sequence to be detected. The oligomer is free in that the oligomer is not conjugated or bonded to the conducting polymer or any secondary substrate. The specimen electrode may be a carbon, metallic or metal-coated crystal electrode. The conducting polymer may be an electropolymerized substance, such as polypyrrole, polythiophene, polyaniline or a derivative thereof. The free oligomer can be an oligonucleotide from about 8 to about 50 mers, and preferably from about 20 to about 30 mers.
The apparatus for detection of DNA hybridization can include means for accumulating a specimen on at least a portion of the polymer coated detection surface of the specimen electrode and means for amperometric detection of the specimen electrode upon accumulation of a specimen on at least a portion of the polymer coated detection surface of the specimen electrode. Means for accumulating a specimen can include a reservoir or other receptacle or volume, and can also include pumps, shunts, tubes and other structure for accumulating a specimen. Means for amperometric detection can include any metering or measuring device or system for amperometric and other electric current-related measurements, which may be digital or analog, and which may be optionally integrated into a computer-based system.
The apparatus for detection of DNA hybridization can further include a reference electrode and means for determining the change in potential of the specimen electrode relative to the reference electrode upon accumulation of a specimen on at least a portion of the polymer coated detection surface of the specimen electrode.
In another embodiment, the invention provides a method for detection of DNA in a test specimen, including the steps of:
providing a specimen electrode with a detection surface;
coating at least a portion of the detection surface with a conducting polymer composition, which composition includes a conducting polymer and a free oligomer complementary to the DNA sequence to be detected;
providing electrical contact to the specimen electrode;
exposing at least a portion of the conducting polymer composition coated detection surface of the specimen electrode to a solution containing the test specimen; and
detecting interactions of the free oligomer and DNA contained in the test specimen.
In the foregoing method, the oligomer is free in that the oligomer is not conjugated or bonded to the conducting polymer or any secondary substrate. The oligomer is complementary to the DNA sequence to be detected if it can be hybridized with the DNA sequence to be detected, even though there may not be a one-for-one correspondence between all base pair members.
In the foregoing method, detecting can be by electrochemical means. One such electrochemical means is determining the change in potential of the specimen electrode relative to a reference electrode upon exposing the specimen electrode to a solution containing the test specimen.
In the method, coating can be by electrochemical deposition of the composition including the conducting polymer and the free oligomer complementary to the DNA sequence to be detected. The electrochemical deposition can be by cyclic voltammetric deposition.
The method can also include the step of electropolymerizing a substance to form the conducting polymer. The substance to be electropolymerized can be a pyrrole, thiophene, aniline or derivative thereof.
In another embodiment, the invention provides an apparatus for detection of nucleic adds in a flowing stream, which apparatus includes a first test electrode with a detection surface, a conducting polymer containing a first dopant coating at least a portion of the detection surface of the first test electrode, means for providing electrical contact to the first test electrode, means for flowing a liquid stream in contact with at least a portion of the detection surface of the first test electrode, and means for detecting nucleic adds in the flowing stream by amperometric detection of adsorption of nucleic adds onto the conducting polymer of the first test electrode. The apparatus can also include a reference electrode in contact with the liquid stream, means for providing electrical contact to the reference electrode, and means for amperometric detection of the first test electrode relative to the reference electrode. The apparatus can further also include a second test electrode with a detection surface, a conducting polymer containing a second dopant coating at least a portion of the detection surface of the second test electrode, means for providing electrical contact to the first test electrode, means for flowing the stream in contact with at least a portion of the detection surface of the second test electrode and means for detecting nucleic acids in the flowing stream by amperometric detection of absorption of nucleic acids onto the conducting polymer of the second test electrode.
In the apparatus for detection of nucleic acids in a flowing stream the conducting polymer can include an electropolymerized substance. The electropolymerized substance can be polypyrrole, polythiophene, polyaniline or a derivative of any of the foregoing. The first and second test electrodes can be carbon, metallic or metal-coated crystal electrodes, and it is possible and contemplated that the first and second test electrodes may differ. Similarly, where a first and second test electrode are employed, the first dopant and second dopant may differ.
In yet another embodiment the invention provides a method for nucleic add detection in a flowing stream, including the steps of:
coating a first test electrode with a conducting polymer containing a first dopant;
providing electrical contact to the first test electrode;
exposing the first test electrode to a flowing steam; and
detecting nucleic acids in the flowing stream by amperometric detection of adsorption of nucleic acids onto the conducting polymer.
The method for nucleic acid detection in a flowing stream can also optionally include the following additional steps:
providing a reference electrode;
providing electrical contact to the reference electrode; and
measuring current changes in the first test electrode relative to the reference electrode.
The method for nucleic acid detection in a flowing stream can further optionally include the following additional steps:
coating a second test electrode with a conducting polymer containing a second dopant;
providing electrical contact to the second test electrode;
exposing the second test electrode to the flowing stream sequentially with exposing the first test electrode and reference electrode to the flowing steam; and
characterizing nucleic acids in the flowing stream by differences in amperometric detection of adsorption of nucleic acids onto the conducting polymer of the second test electrode and the first test electrode.
In this method for nucleic acid detection in a flowing stream both the first and second dopant can be anionic, and either can be a source of nitrate ion or an oligonucleotide. Different dopants can be utilized to yield different detection signals using the same specimen, so that the specimen may be characterized by differences in the detection signals between the first and second test electrodes.
The step of coating the test electrodes includes coating by electrochemical deposition of the conducting polymer and the dopant. The electrochemical deposition can be by cyclic voltammetric deposition. This method can also include the step of electropolymerizing a substance to form the conducting polymer. The substanced to be electropolymerized can be pyrrole, thiophene, aniline or derivative thereof.
A primary object of the present invention is to provide methods and compositions for the rapid, label-free electrochemical detection of nucleic acids, including oligonucleotides, DNA and RNA.
Another object of the present invention is to provide methods and compositions for label-free electrochemical detection of DNA hybridization.
Another object of the present invention is to provide methods and compositions for electrochemical detection of DNA hybridization utilizing an electropolymerized film wherein the sole dopant is the oligonucleotide probe.
Another object of the invention is to provide methods and compositions for electrochemical detection of DNA hybridization wherein distinct hybridization peak signals are observed in the presence of both complementary and non-complementary DNA sequences, with the peaks being of opposite direction for complementary and non-complementary DNA sequences.
Another object of the invention is to provide methods and compositions for electrochemical detection of nucleic acids utilizing conducting polymer electrodes using flow injection analysis.
Another object of the invention is to provide methods and compositions utilizing polymeric films, such as polypyrrole, polythiophene or polyaniline, for flow injection analysis, such as in chromatography or electrophoresis systems, wherein detection of nucleic acids results from rapid adsorption and desorption of nucleic acids onto the film during the passage of the sample over a film-coated electrode.
A primary advantage of the present invention is that, in one embodiment, it provides a fast, sensitive, label-free and universal method for the detection of oligonucleotides, DNA and RNA.
Another advantage of the present invention is that, in one embodiment, it provides methods and compositions for detection of nucleic acids utilizing a polymeric film, such as polypyrrole, polythiophene or polyaniline, wherein a nucleic acid is the sole dopant within the polymeric film, and serves as the sole charge compensating counter ion during film formation.
Another advantage of the present invention is that it provides methods and compositions for detection of nucleic acids and hybridization events utilizing a polymeric film, such as polypyrrole, polythiophene or polyaniline, wherein the detection scheme can discriminate against oxidizable non-nucleic acid species present in biological samples.
Another advantage of the present invention is that it provides methods and compositions which may be employed as biomaterials for genoelectronic devices, composite materials, bioactive interfaces, flow systems, microscale separations, xe2x80x9con-chipxe2x80x9d devices and similar biomaterial-based devices, for use in genetic analysis, diagnostic applications, microscale separations, on-line PCR amplification, batch injection analysis, hybridization analysis and the like.
Other objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.